Fgf9 regulates bone marrow mesenchymal stem cell fate and bone-fat balance in osteoporosis by PI3K/AKT/Hippo and MEK/ERK signaling

Bone-fat balance is crucial to maintain bone homeostasis. As common progenitor cells of osteoblasts and adipocytes, bone marrow mesenchymal stem cells (BMSCs) are delicately balanced for their differentiation commitment. However, the exact mechanisms governing BMSC cell fate are unclear. In this study, we discovered that fibroblast growth factor 9 (Fgf9), a cytokine expressed in the bone marrow niche, controlled bone-fat balance by influencing the cell fate of BMSCs. Histomorphology and cytodifferentiation analysis showed that Fgf9 loss-of-function mutation (S99N) notably inhibited bone marrow adipose tissue (BMAT) formation and alleviated ovariectomy-induced bone loss and BMAT accumulation in adult mice. Furthermore, in vitro and in vivo investigations demonstrated that Fgf9 altered the differentiation potential of BMSCs, shifting from osteogenesis to adipogenesis at the early stages of cell commitment. Transcriptomic and gene expression analyses demonstrated that FGF9 upregulated the expression of adipogenic genes while downregulating osteogenic gene expression at both mRNA and protein levels. Mechanistic studies revealed that FGF9, through FGFR1, promoted adipogenic gene expression via PI3K/AKT/Hippo pathways and inhibited osteogenic gene expression via MAPK/ERK pathway. This study underscores the crucial role of Fgf9 as a cytokine regulating the bone-fat balance in adult bone, suggesting that FGF9 is a potentially therapeutic target in the treatment of osteoporosis.


Figure S6 .
Figure S6.Analysis of DEGs and mRNA level of bone-fat related genes.

Figure S7 .
Figure S7.Quantitative results of protein levels from Figure 6.

Figure S1 .
Figure S1.Fgf9 in bone marrow clusters.(A) UMAP visualization of all bone marrow cells from the integrated analysis, color-coded based on the clusters.(B) Distribution of Fgf9 in bone marrow stroma cell clusters.(C) Expression of Fgf9 and Fgfrs in bone marrow clusters.The size of dots represents the percentage of expression; red and blue represent the level of scaled mean expression.(D) Immunofluorescence staining of S100A4 in Fibroblast-like cells and BMSCs.(E) S100A4 + cell ratio in Fibroblast-like cells and BMSCs.Data are analyzed by Student's t-test and shown as boxplots (median ± interquartile range).Scale bars represent 300 μm.

Figure S2 .
Figure S2.Fgf9 S99N mutation mitigates BMAT accumulation in adult mice.(A) Statistical analysis of femur adipocytes area in 4-month-old wt and het mice.(B) H&E staining of rBMAT in tibiae from 4-month-old male wild-type and heterozygous mice.(C) Statistical analysis of rBMAT adipocyte number in 4-month-old wt and het mice.

( D )
H&E staining of cBMAT in tibiae from 4-month-old male wt and het mice.(E) Statistical analysis of cBMAT adipocytes area in 4-month-old wt and het mice.Data are analyzed by Student's t-test and shown as boxplots (median ± interquartile range), n = 4 mice in each group.Scale bars represent 200 μm.

Figure S3 .
Figure S3.Fgf9 S99N mutation involves in bone-fat balance.(A-D) Bone volume (BV), Bone surface (BS), Percent bone volume (BV/TV), and Connectivity (Conn) were determined by micro-CT analysis.(E) Micro-CT images of the lateral-view of trabecular bone of the metaphyseal region (above) and top-view cortical bone (below),

Figure S5 .
Figure S5.Differentiation of OE-Ctrl/OE-Fgf9 BMSCs in vivo.(A) mRNA expression level of Fgf9 increased dramatically after transfection.n = 3 biological replicates over three independent experiments.(B) After subcutaneous injection for 5 weeks, smaller magnifications of H&E staining, n = 3 mice.Data are analyzed by Student's t-test and shown as boxplots (median ± interquartile range).Scale bars represent 200 μm.